Bacillus subtilis DB430饋料式發酵及基因重組蛋白表現之研究

Abstract

過去的研究得知，Bacillus subtilis有很多的優點，並適合基因重組蛋白質產品生產用。本論文研究目的是為了提高Bacillus subtilis發酵產物的產量，在已知最佳的碳源（葡萄糖）及氮源（酵母抽出物）搭配下去調控其他發酵條件，使達到工業化生產水準。我們同時也進行菌株突變以獲得更適合之菌種，並改造質體使其在菌株內的穩定性上升。從搖瓶式發酵培養枯草桿菌的實驗中得知氮源（酵母抽出物）的消耗速率為每百分之一酵母抽出物使菌體O.D.600上升8、最佳的鹽類添加、及最佳的起始濃度為NaCl 0.3g/l、 K2HPO4 0.4g/l、 CaCl20.2g/l、 MgSO4 0.5g/l、 FeSO4 30mg/l、 MnSO4 20mg/l。進一步在發酵槽中用上述之條件進行大量培養，並以電腦控制葡萄糖、酵母抽出物及各種鹽類的進料速率。由於枯草桿菌為好氧菌，其對氧濃度的變化十分敏感，因此我們根據D.O.數值來調配進氣量及其進氣含氧的濃度。當D.O.值保持在10~20%之間，在精密的控制下件進行饋料培養，可得到最高的菌體濃度，其OD600可達154，菌體乾重(biomass)可達54 g/l。另一方面，為了保護重組DNA所表現的蛋白質不被破壞，因此構築recE的突變株。我們取得Kmr及Cmr基因並將兩基因分別插入正常recE基因中使其失活。再把失活recE送入DB430中行double crossover recombinant，將正常的recE基因換掉，得到含Cmr的recE突變株。此外本研究中外來表現蛋白質為口蹄疫病毒之表面蛋白VP1及VP3，分子量均為25kDa。從蛋白質電泳圖得知VP1及VP3均有表現，其中VP3在DB430中經饋料式發酵可得到每公升1.4克的蛋白表現。另外已知Restriction - Modification system有助質體穩定，現已從 E coli RY13取得EcoRI之R-M system，轉殖到質體的工作目前尚在進行中。

Bacillus subtilis has many advantages for biotechnology that including nonpathogenic, easy to cultivate, store and secrete protein. Therefore, it has been used to product the recombinant DNA proteins. The goal of this study is to improve the biomass as well as recombinant proteins production ability in Bacillus subtilis. In order to obtain the high cell density, the optimization of fed-batch culture was studied. The experiment was carried out by using glucose as carbon source and yeast extract as nitrogen source. The parameters of control fermentation have been examined which includes how to manipulate Carbon and nitrogen sources feeding, agitation and aerate, as well as trace elements supply. The results showed that the optimal glucose concentration is 0.5% ( w/v ), and the consumption tare of yeast extract is 1%( w/v ) when O.D.600 value increased 8. When Bacillus subtilis DB430 grew in 2.5L fermenter at 37℃ and 700 rpm of stir rate and used 50% of oxygen, we can got the highest cell density ( O.D.600 value is 154 ) and the cell dry weight ( biomass is 54 g/l ). The highest expression of recombinant VP3 protein was1.4 g/l in fed-batch culture at 37℃, 700 rpm of stir rate. To protect the recombinant protein, a recE mutant was also construct in Bacillus subtilis DB430.